Recombinant vaccine technology has the potential to provide a rapid response to emerging influenza virus infections with pandemic potential, since it does not rely on the derivation of vaccine viruses with high growth in eggs. The VLP platform is particularly attractive for vaccines against highly pathogenic zoonotic influenza virus strains, since it does not require the use of live virus in any stage of the manufacturing process. Furthermore, VLPs present the key antigenic targets of influenza virus, HA and NA, in a native-like conformation similar to the intact virion. Studies in animal models also provided evidence for generation of cross-clade protective antibodies following H5N1 VLP (but not rHA) immunization (4
The current study was conducted with immune sera from a subset of participants in the first human phase I/II trial of an A/Indonesia/05/2005 (H5N1) VLP vaccine candidate. The vaccine was generally well tolerated, and after two vaccinations, HAI seroconversion rates (SCRs) of 40%, 57%, and 61% and MN SCRs of 39%, 52%, and 76% were achieved in the 15-, 45-, and 90-μg groups, respectively. Cross-reactivity against other H5N1 clade 2 subtypes was also demonstrated with high-responder sera. GFPDL analyses (A/Indonesia/5/2005; HA/NA) revealed broad epitope repertoires recognized by immune sera from the two higher-antigen-dose groups, which included large segments spanning the RBD in the HA1 globular head and the NA C terminus. We identified an immunodominant epitope in the HA2 domain in preimmune sera that was significantly boosted following vaccination. A similar epitope was identified in our previous studies with H5N1 A/Vietnam/1203/2004 vaccine trials (16
). This HA2 epitope sequence is highly conserved (98%) within H5N1 and seasonal H1N1 strains and therefore may be present due to preexisting immunity from either vaccination or exposure to seasonal H1N1 strains. Since reactivity to this HA2 epitope was also boosted with the 15-μg VLP dose, which had no significant virus-neutralizing activity, it is unlikely that antibodies against this HA2 epitope contribute to protection.
Binding of antibodies to properly folded oligomeric H5N1 rHA1(1-320) protein was significantly higher than that to monomeric rHA1(28-320) for all sera obtained following high-dose vaccinations. The oligomeric/monomeric HA1 binding ratio correlated with virus neutralization titers. Neuraminidase inhibition ratios of above 2 (post-second vaccination/prevaccination) were detected in sera from the high-dose groups and correlated with binding to an NA C-terminal epitope that is located close to the sialic acid binding site.
The HAI titers and cross-clade reactivity of sera in the human trial are in agreement with the preclinical studies involving ferrets and a similar VLP vaccine product (30
). The H5N1 VLP vaccine was immunogenic, and the HAI titers were comparable to those reported with an inactivated egg-derived H5N1 (A/Vietnam/1203/04) vaccine previously licensed in the United States (1
). Furthermore, we measured heterologous neutralization of other clade 2 subtypes not found after vaccination with the egg-based inactivated subunit A/Vietnam vaccine unless combined with oil in water adjuvants (16
). A plant-based H5N1 VLP vaccine was also reported to elicit cross-reactive neutralizing antibodies in animals and humans (23
). This difference could also reflect a difference in the immunogenicities of A/Indonesia/05/2005 HA and A/Vietnam/1203/04, as previously observed in animal experiments (4
). More recently, a VLP vaccine against the pandemic H1N1 strain was evaluated in the Mexican population and was found to be safe and immunogenic (27
The role of oligomeric HA in eliciting protective antibodies is an important finding. There is growing evidence that oligomeric vaccines present epitopes that are specific to virion spikes and that are absent on monomeric proteins. Furthermore, antibodies that bind preferentially (with high avidity) to oligomeric compared with monomeric HA proteins were reported to have broader cross-reactivity (31
). Therefore, several recombinant influenza virus and HIV vaccines were designed to include trimerization domains in order to elicit broadly neutralizing antibodies (3
). Since the H5N1 VLP vaccine used in the current trial naturally presents multimeric HA and NA to the immune system, it was important to determine if the human immune sera contained antibodies that bind differentially to oligomeric versus monomeric structures. In earlier studies we have expressed recombinant HA1 proteins from multiple influenza virus strains that were enriched for oligomeric or monomeric HA structures. We previously demonstrated that oligomeric rHA1 proteins were highly immunogenic and induced superior protective immunity against homologous and heterologous strains compared with monomeric HA1 or HA0 recombinant proteins (references 20
and unpublished data). In the current study, we demonstrated that the H5N1 VLPs generated antibodies that preferentially bound to oligomeric A/Indonesia/5/2005 rHA1(1-320) compared with monomeric rHA1(28-320) protein (). Furthermore, a positive correlation between the oligomeric/monomeric HA1 binding ratios and the in vitro
neutralization titers for individual postvaccination human sera was demonstrated. In concordance, we found that the oligomeric/monomeric binding ratios of human immune sera from egg-based H5N1 subunit vaccination (A/Vietnam/1203/04, SP) (1
) were around 1, with no clear preference for oligomeric HA1 binding (data not shown).
Neuraminidase-neutralizing antibodies elicited by vaccines may provide added value to the protective immunity against both seasonal and avian influenza viruses. In the case of traditional inactivated subunit vaccines, the neuraminidase content is not monitored, and the fraction of functional protein may vary considerably with the manufacturing process (7
). In contrast to the inactivated influenza virus vaccines, the VLP platform presents NA in its native tetrameric form, thereby increasing the opportunity to elicit protective antibodies against this important viral membrane protein. The reactivity of the antibodies against the NA C terminus, demonstrated in the current VLP vaccine trial, is likely to interfere with the binding of NA to sialic acid, which is required for its enzymatic activity and release of virions from infected cells.
In summary, our data provide the first insight into the quality of the epitope repertoire and avidity of the antibodies elicited by H5N1 VLP vaccine in humans. The increased binding to oligomeric HA and the C terminus of NA may contribute to the broader cross-reactivity of the immune sera seen in subjects administered VLP vaccines. However, no clear dose sparing was provided by the VLP platform compared with the inactivated H5N1 vaccine. Future trials should determine if a VLP-adjuvant combination could improve immunogenicity and dose response.